In this post-genomic era, chromosome engineering is increasing its technological importance. Techniques for development of living organisms with chromosomes carrying desired modifications such as gene disruption, foreign gene insertion and mutagenesis are extremely important in various fields such as molecular biology, basis medicine and agricultural engineering. Various techniques for chromosome modification have been developed to date, including the λ-red recombination system (Non-patent Document 1), the Cre/loxP system (Non-patent Document 2), the Flp/FRT system (Non-patent Document 3) and a system using meganuclease (Non-patent Document 4). These methods have their own characteristics and have drawbacks such as the foreign inserts remaining after chromosome modification, and the difficulty associated with determining the optimal conditions for enzyme expression. Moreover, all these methods not only require specific enzymes and sequences but also are time-consuming and troublesome because two or more steps are generally required to obtain the modified target strain.
Chromosome modification in S. cerevisae and C. albicans using URA3 (orotidine 5′-phosphate decarboxylase gene) as a selectable marker gene is known (FIG. 1) (Non-patent Documents 5 and 6). FIG. 1 schematically illustrates the URA3 recycling method previously developed in S. cerevisae and C. albicans. In this method, a modification fragment containing repeated sequences is inserted (FIG. 1). The target DNA region of a chromosome (the parental strain in FIG. 1) is replaced by the DNA fragment carrying a negative selectable marker gene and hence deleted. URA3 as the selectable marker is flanked by repeated sequences (the sequences upstream and downstream of the URA3 selectable marker gene), and the inserted modification fragment enclosed with a dotted line contains the repeated sequences. The URA3 selectable marker gene allows both positive and negative selections. While prototrophs are selected in a uracil-deficient medium, auxotrophs are selected in a medium containing 5-fluoroorotic acid (hereinafter referred to as 5-FOA) in which uracil prototrophs are unable to grow because 5-FOA is an analogue of a uracil precursor and is known to be converted to fluorouracil, a strong growth inhibitor for yeasts (Non-patent Document 7).
Non-patent Document 1: Wong, Q. N. et al. Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli. Nucl. Acids Res. 33, e59 (2005).
Non-patent Document 2: Ghosh, K. & DuyneG. D. Cre-loxP biochemistry. Methods 28, 374-383 (2002).
Non-patent Document 3: Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97, 6640-6645 (2000).
Non-patent Document 4: Posfai, G., Kolisnychenko, V., Bereszki, Z. & Blattner, F. R. Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome. Nucl. Acids Res. 27, 4409-4415 (1999).
Non-patent Document 5: Langle-Rouault, F. & Jacobs, E. A method for performing precise alterations in the yeast genome using are cyclable selectable marker. Nucl. Acids Res. 23, 3079-3081 (1995).
Non-patent Document 6: Wilson, R. B., Davis, D., Enloe, B. M.& Mitchell, A. P. A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions. Yeast 16, 65-70 (2000).
Non-patent Document 7: Grimm, C., Kohli, J., Murray, J. & Maundrell, K. Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol. Gen. Genet. 215, 81-86 (1998).